Traditional thinking is that nuclear ER? modulates the genes that are essential to breast cancer development. Although this is certainly true, additional actions of estrogen at locations outside the nucleus contributes to both transcriptional and non-transcriptional effects. In our initial grant period, we established important new principals governing the localization, signaling, and cell functions of ER? at the plasma membrane. Further, we published the first data implicating mitochondrial ER? in breast cancer cells, contributing to cell survival after exposure to radiation. We now propose studies to clarify important extra-nuclear ER functions in breast cancer. We identified a conserved 9 amino acid motif in ER?, ER?, and both androgen and progesterone receptors that dictates palmitoylation, resulting in membrane localization of the sex steroid receptors. We now propose to isolate and characterize the palmitoylacyltransferase (PAT) protein(s) that palmitoylate the steroid receptors, and palmitoylation-modulating proteins (e.g.-Hsp27), each resulting in membrane localization. These proteins could be therapeutic targets in breast cancer. In specific aim 2, we propose a novel basis for tamoxifen resistance involving ER? in mitochondria. Tam-sensitive MCF7 and T47D cell lines respond to Tam with strong reactive oxygen species (ROS) formation from mitochondria, triggering PKC? and JNK activation, recruitment of Bax and Bak to the mitochondria, and membrane potential decrease. This results in apoptosome formation to trigger effector caspase activation and apoptosis. In Tam-sensitive cells, Tam engages ER? in the mitochondria as an antagonist, decreasing MnSOD activity that is important to ROS generation and subsequent death signaling. In Tam-resistant MCF7 and T47D cells, Tam engages ER? in the mitochondria as an agonist, stimulating MnSOD activity that quenches ROS formation. MnSOD knockdown with siRNA reverts Tam resistant cells to sensitive cells where Tam now causes substantial apoptosis. These studies identify novel mechanisms of Tamoxifen in resistance/sensitivity, mediated differentially through mitochondrial ER?. Whether these mechanisms underlie aromatase inhibitor resistance will also be determined. In human breast cancer specimens, it is unclear whether plasma membrane ER? contributes to tumor development or outcome. We propose in the final aim to use a modified Allred score, comparing membrane and nuclear ER? expression in 300 breast tumors. We will correlate membrane and nuclear ER? Allred expression scores to many clinical parameters, including tumor type, metastasis, response to various therapies (including tamoxifen), mortality, and BRCA1 mutation status from 15 years data. The clinical significance is to understand new actions of estrogen and tamoxifen that are important to breast cancer biology, possibly targeting membrane or mitochondrial ER for preventing or treating breast cancer.